The completion of Human Genome Project has opened the door to a flood of postgenomic applications that rely on genomic sequences. However DNA sequence information alone is not able to predict gene expressions, co- and post-translational modifications and phenotype or multigenic phenomena such as drug administration, cell cycle, oncogene, aging, stress and disease. The understanding of probably half a million human proteins and their relationship to physiological or disease conditions is still a long way away. Proteome is a new fundamental concept, which has recently emerged, that should play a significant role in the battle to unravel biochemical and physiological mechanism of complex multivariate diseases at the functional molecular level. Proteomics can be defined as the qualitative and quantitative comparison of proteomes under different conditions to further unravel biological processes. Proteomics is becoming an important topic in the effort to understand gene regulations and the changes in protein profiles in cellular systems either in physiological conditions or disease conditions. Although still in its infancy, proteomics already promises revolutionary changes in biological and medical sciences.
Proteome studies have developed and continue to be dependent upon the core technology of multidimensional separation techniques, such as 2-D gel electrophoresis (2-DE), and more recently, combined chromatography techniques (e.g. LC-LC). Despite the increased popularity of the latter, 2-D gel electrophoresis remains one of the most frequently chosen methods of choice due to its ability to separate complex mixtures of proteins and to follow multigenic phenomena at the level of whole cells, tissue, and even whole organisms.
2-DE is a method for the separation and identification of proteins in a sample by displacement in 2 dimensions oriented at right angles to one another. Isoelectric focusing (IEF) is used in the first dimension, which separates proteins according to their isoelectric points (pIs). The pI is the pH at which an amphoteric substance is neutral charged. When pH is higher than the pI of a substance, it will be negative charged and vise versa. When a protein is placed in a medium with a pH gradient and subjected to an electric field, it will initially move toward the electrode with the opposite charge. As it migrates, its net charge and mobility will decrease and the protein will slow down. Eventually, the protein will arrive at the point in the pH gradient equal to its pI, where it becomes neutral and stop migrating. If the protein should happen to diffuse to a region of lower (higher) pH, it will become positively (negatively) charged and be forced back toward the cathode (anode) by the electric field. In this way, proteins are focused into sharp bands in the pH gradient at their individual characteristic pI values. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is the method for the second dimension separation, in which protein separation is based on molecular weight. SDS binds to all proteins, and the net effect is that proteins migrate with a uniform negative charge-to-mass ratio. The pores of polyacrylamide gel sieve proteins according to size (i.e. molecular weight). Smaller proteins move faster through the gel under the electric field than larger proteins, thus achieve molecular sieving. Compared to 1-D electrophoresis based separation methods, 2-D strategy allows the sample to separate over a larger area, increasing the resolution of each component. E.g. unlike PAGE 1-D separation, which can only separate about 100 bands, 2-D gel based separations can analyze samples with much higher complexity (e.g. common 2-D runs can separate thousands of spots).
When first developed, 2-DE utilized carrier ampholyte (CA) to generate pH gradient for the first dimension IEF separation. However there are many disadvantages associated with CA-IEF. Firstly, equilibrium CA-IEF cannot be achieved because of cathodic drift, which means that pH gradient moves towards the cathode end with prolonged focusing time. Secondly, reproducibility of pH gradient is also influenced by the batch-to-batch variability of CA preparations, and by the sources where the CA is obtained. Therefore, the exchange of 2-D gel data between laboratories has been a major problem because of spatial irreproducibility between 2-D gels generated by the CA-IEF. The problems of pH gradient instability and irreproducibility were solved by the introduction of immobilize pH gradient (IPG) for IEF. Certain chemicals, co-polymerized with the acrylamide matrix, generate a much stable pH gradient. The advantages of IPG include the elimination of cathodic drift, an enhancement in reproducibility. Also, the introduction of IPG has greatly improved resolution, especially with the usage of narrow-range IPGs. This advancement makes 2-D gel electrophoresis a core technology of proteome analysis, facilitating spot identification by peptide fingerprinting, amino acid composition, analysis peptide sequencing and mass spectrometry analysis.
However, there are still many disadvantages in this 2-DE technique, which may limit its usage in proteomics researches. The disadvantages of 2D gel electrophoresis include labor-intensive (sample treatment, gel preparing, staining), time consuming (usually takes multiple days to complete one analysis), and not readily automated (need constant human attention). Low throughput is another important factor influencing the cost effectiveness of conventional approaches to proteome analysis. Despite numerous refinements in electrophoretic techniques over the past decade, the above disadvantages associated with the 2-D gel electrophoresis still exist. The process is still tedious and inefficient and the time required to prepare, load, separate and visualize complex mixtures of proteins on conventional 2-D gels is still substantial. To meet the needs of proteomic research, new technologies still need to be developed with the following features: 1) must have increased resolving power; 2) must have higher sensitivity and speed; 3) must be automated and easy to use; 4) must have the ability to perform high throughput analysis and 5) better have the potential to couple with some other techniques such as MS.
Improvements in speed, sensitivity, resolution and automation can be achieved by using capillary electrophoresis (CE), which offers many advantages over traditional gel electrophoresis for the separation of a wide variety of molecules. Multiplexed capillary electrophoresis, which utilizes parallel capillary tubes simultaneously, also provides enhanced throughput. CE on microchips is an emerging new technology that promises to lead the next revolution in chemical analysis. Over the past decade, the field of microfabrication of analytical devices has grown from an esoteric technique to a recognized technique with commercially available systems. It has the potential to simultaneously assay hundreds of samples in a short time, and also is easier to interface with MS than traditional CE. Isoelectric focusing on microfabricated devices using either natural pH gradient (Analytical Chemistry, 2000, 72, 3745-3751) or ampholyte-generated pH gradient (Electrophoresis, 2002, 23, 3638-3645) has been reported. These resemble the first dimension separation in traditional 2-D gel electrophoresis. On the other hand, efforts have been made to separate proteins based on their sizes in microfludic channels (Proc. Natl. Acad. Sci. USA, 1999, 96, 5372-5377). However these applications are based on single-channel devices, and therefore only provide 1-D separation. Whitesides et al. have constructed a chip device by PDMS (polydimethylsiloxane), on which 2D gel electrophoresis has performed (Analytical Chemistry, 2002, 74, 1772-1778). The first dimension is isoelectric focusing and the second dimension is SDS gel electrophoresis. However, this device is not automated in part because after the first dimension separation, the device has to be manually dissembled and then re-assembled to physically connect the first dimension to the second dimension channels.
These above-mentioned microchip-based studies used fluorescence detection, which required labeling of the protein with dyes, which will change the properties of the sample (e.g. pI and molecular weight). UV (ultraviolet) absorption detection is more useful because of its ease of implementation and wider applicability, especially for the deep-UV (200-220 nm) detection of organic and biologically important compounds. In a UV detection system, a section of capillary tube or microfabricated channel is irradiated with a UV light source. A photodetector detects the light that passes through the tube. When a UV absorbing sample component passes through the irradiated portion of the capillary tube, the photodetector detects less passed light (indicating absorbance). In this way an electropherogram, a plot of absorbance versus time, can be produced. Photodiode arrays (PDA) are used in many commercial CE and HPLC (high performance liquid chromatography) systems for providing absorption spectra of the analytes in real time. Transmitted light from a single point in a flow stream is dispersed by a grating and recorded across a linear array. Yeung et al., in PCT Application WO 01/18528A1, disclosed a multiplexed, absorbance-based capillary electrophoresis system for analyzing multiple samples simultaneously. The use of UV absorbance eliminates the need for protein staining, which is a major contributor for the long time used in traditional 2-D gel electrophoresis.
The primary objective of this invention is to fulfill the above described needs in proteomics study with an improved multiplexed, absorbance-based system.
A further objective is an automated alternative for the 2-D gel electrophoresis, which will provide higher speed, higher sensitivity, higher resolution and higher throughput.
A still further objective is a chip-based microfludic device that at one dimension can perform isoelectric focusing, and at another dimension can perform size-based protein separation.
A still further objective is to include separation mechanisms and the type of information recorded similar to 2-D gel electrophoresis. A still further objective is to include UV absorbance detection.
One or more of these and/or other objectives will become apparent from the specification and claims that follow.